Fluorescent semiconductor nanoparticles (quantum dots, QDs) exhibit several unique optical and spectroscopic properties that are not observed in their bulk parent materials or at the molecular scale. See References 1-5. Quantum dots made of CdSe, CdS, InAs, and InP cores have tunable size-dependent broad absorption, with high extinction coefficient, and size-dependent narrow Gaussian emission profiles. See References 4, 6, and 7. CdSe-based nanoparticles, in particular, exhibit remarkable resistance to chemical and photo-degradation and a high two-photon action cross-section. See References 8-11. For a wide range of bio-inspired applications, the optical and spectroscopic properties exhibited by luminescent QDs are unmatched by organic dyes and fluorescent proteins, which has spurred significant interest in developing QDs as fluorescent platforms and markers to increase our understanding of a variety of biological processes, ranging from sensing to the tracking of intracellular protein movements and interactions. See References 8 and 10-19. An important requirement for the effective integration into biotechnology is, however, the ability to access stable, water-soluble reagents that can be manipulated under a wide variety of conditions (basic and acidic pHs, high concentrations of electrolytes and in the presence of reducing agents) and chemistries; the latter should allow straightforward and controllable coupling to biomolecules such as proteins, peptides and nucleic acid oligomers. See References 10, 11, 15, 19, and 20.
Highly luminescent quantum dots are reproducibly prepared with narrow size distribution and high fluorescence quantum yields by reacting organometallic precursors at high temperature in coordinating solutions. See References 3, 4, 6, and 21-28. These QDs are typically capped with hydrophobic molecules (trioctyl phosphine (TOP), trioctyl phosphine oxide (TOPO), and alkylamines) and are dispersed in non-polar organic solvents. Post-synthetic surface modifications are thus required to render them hydrophilic and biocompatible. An established strategy for preparing water-soluble QDs relies on replacing the native hydrophobic cap with bifunctional hydrophilic ligands that combine metal-chelating anchors onto the nanocrystal surfaces and hydrophilic modules that promote aqueous compatibility. See References 29-35. Two inherent properties of the ligands greatly influence the stability of the hydrophilic QDs: 1) the coordination affinity of the anchoring groups to the inorganic surface; and 2) the method for achieving aqueous compatibility of the nanocrystals, i.e., whether it is driven by electrostatic repulsions between lateral charges, or results from strong ligand affinity to the surrounding buffer. See References 17 and 17-20.
Ligands presenting two and four thiol anchoring groups, such as polyethylene glycol-appended with one or two dihydrolipoic acid groups, DHLA-PEG and bis(DHLA)-PEG, have been shown to provide greatly enhanced stability over a wide range of biological conditions compared to their monothiol-ligands, while significantly reducing non-specific interactions. See References 30-32, 36, and 37. This result has been attributed to the higher coordination onto the nanocrystal surface afforded by the multi-thiols (compared to their monothiol-ligands), which essentially shifts the coordination equilibrium and decreases the dissociation rate of the ligand from the QD surfaces. See References 29-31. Enhanced Au nanoparticle stability afforded by these multidentate ligands is also well documented. See References 32, 36, and 38-42. However, reduction of the lipoic acid (LA) groups to DHLAs is required for effective capping of QDs. See References 29, 36, and 43. To date, the requisite reduced form of lipoic acid used for the phase transfer has been prepared via prior chemical reduction of the dithiolane ring under strong reducing conditions using NaBH4. See References 37 and 44. Alternative reduction route relying on electrochemical methods has been reported. See References 45 and 46. While effective, this process imposes limitations with respect to tolerating other functional groups on the LA-based ligands and introduces an extra processing step with specific requirements. For instance, we found that NaBH4-reduction can alter the integrity of certain functional groups such as the terminal azide on LA-PEG-N3. This route also requires careful storage of the reduced DHLA-based ligands under inert atmosphere to avoid re-oxidation of the dithiol back to a disulfide. See References 29, 30, and 44.
A previous study by Sander and co-workers reports that the cyclic disulfide core of lipoic acid has a well defined absorption at ˜350 nm and excitation at this wavelength produces a relatively long-lived (τ˜0.1 μs) triplet state, which can be converted into DHLA. See Reference 47. The reported yields for this process were moderate (up to ˜25%).